Pesticidal proteins and methods for their use

ABSTRACT

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a toxin polypeptide are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated toxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed, and antibodies specifically binding to those amino acid sequences. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:50-96, or the nucleotide sequence set forth in SEQ ID NO:1-47, as well as variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.13/655,664 filed Oct. 19, 2012, which is a continuation of U.S.application Ser. No. 12/713,239, filed Feb. 26, 2010, now U.S. Pat. No.8,318,900, issued Nov. 27, 2012, which claims the benefit of U.S.Provisional Application Ser. No. 61/156,301, filed Feb. 27, 2009, thecontents of which are herein incorporated by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“APA062USO1N_SEQLIST.txt”, created on Oct. 15, 2012, and having a sizeof 698 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These proteins and thenucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a Gram-positive spore forming soil bacteriumcharacterized by its ability to produce crystalline inclusions that arespecifically toxic to certain orders and species of insects, but areharmless to plants and other non-targeted organisms. For this reason,compositions including Bacillus thuringiensis strains or theirinsecticidal proteins can be used as environmentally-acceptableinsecticides to control agricultural insect pests or insect vectors fora variety of human or animal diseases.

Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensishave potent insecticidal activity against predominantly Lepidopteran,Dipteran, and Coleopteran larvae. These proteins also have shownactivity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga, andAcari pest orders, as well as other invertebrate orders such asNemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson(1993) The Bacillus Thuringiensis family tree. In Advanced EngineeredPesticides, Marcel Dekker, Inc., New York, N.Y.) These proteins wereoriginally classified as CryI to CryV based primarily on theirinsecticidal activity. The major classes were Lepidoptera-specific (I),Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III),Diptera-specific (IV), and nematode-specific (V) and (VI). The proteinswere further classified into subfamilies; more highly related proteinswithin each family were assigned divisional letters such as Cry1A,Cry1B, Cry1C, etc. Even more closely related proteins within eachdivision were given names such as Cry1C1, Cry1C2, etc.

A new nomenclature was recently described for the Cry genes based uponamino acid sequence homology rather than insect target specificity(Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In thenew classification, each toxin is assigned a unique name incorporating aprimary rank (an Arabic number), a secondary rank (an uppercase letter),a tertiary rank (a lowercase letter), and a quaternary rank (anotherArabic number). In the new classification, Roman numerals have beenexchanged for Arabic numerals in the primary rank. Proteins with lessthan 45% sequence identity have different primary ranks, and thecriteria for secondary and tertiary ranks are 78% and 95%, respectively.

The crystal protein does not exhibit insecticidal activity until it hasbeen ingested and solubilized in the insect midgut. The ingestedprotoxin is hydrolyzed by proteases in the insect digestive tract to anactive toxic molecule. (Möfte and Whiteley (1989) Microbiol. Rev.53:242-255). This toxin binds to apical brush border receptors in themidgut of the target larvae and inserts into the apical membranecreating ion channels or pores, resulting in larval death.

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Aside from delta-endotoxins, there are several other known classes ofpesticidal protein toxins. The VIP1/VIP2 toxins (see, for example, U.S.Pat. No. 5,770,696) are binary pesticidal toxins that exhibit strongactivity on insects by a mechanism believed to involve receptor-mediatedendocytosis followed by cellular toxification, similar to the mode ofaction of other binary (“A/B”) toxins. A/B toxins such as VIP, C2, CDT,CST, or the B. anthracis edema and lethal toxins initially interact withtarget cells via a specific, receptor-mediated binding of “B” componentsas monomers. These monomers then form homoheptamers. The “B”heptamer-receptor complex then acts as a docking platform thatsubsequently binds and allows the translocation of an enzymatic “A”component(s) into the cytosol via receptor-mediated endocytosis. Onceinside the cell's cytosol, “A” components inhibit normal cell functionby, for example, ADP-ribosylation of G-actin, or increasingintracellular levels of cyclic AMP (cAMP). See Barth et al. (2004)Microbiol Mol Biol Rev 68:373—402.

The intensive use of B. thuringiensis-based insecticides has alreadygiven rise to resistance in field populations of the diamondback moth,Plutella xylostella (Ferré and Van Rie (2002) Annu. Rev. Entomol.47:501-533). The most common mechanism of resistance is the reduction ofbinding of the toxin to its specific midgut receptor(s). This may alsoconfer cross-resistance to other toxins that share the same receptor(Ferré and Van Rie (2002)).

SUMMARY OF INVENTION

Compositions and methods for conferring pest resistance to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for toxinpolypeptides, vectors comprising those nucleic acid molecules, and hostcells comprising the vectors. Compositions also include the polypeptidesequences of the toxin, and antibodies to those polypeptides. Thenucleotide sequences can be used in DNA constructs or expressioncassettes for transformation and expression in organisms, includingmicroorganisms and plants. The nucleotide or amino acid sequences may besynthetic sequences that have been designed for expression in anorganism including, but not limited to, a microorganism or a plant.Compositions also comprise transformed bacteria, plants, plant cells,tissues, and seeds.

In particular, isolated nucleic acid molecules corresponding to toxinnucleic acid sequences are provided. Additionally, amino acid sequencescorresponding to the polynucleotides are encompassed. In particular, thepresent invention provides for an isolated nucleic acid moleculecomprising a nucleotide sequence encoding the amino acid sequence shownin any of SEQ ID NO:50-96, or a nucleotide sequence set forth in any ofSEQ ID NO:1-47, as well as variants and fragments thereof. Nucleotidesequences that are complementary to a nucleotide sequence of theinvention, or that hybridize to a sequence of the invention are alsoencompassed.

The compositions and methods of the invention are useful for theproduction of organisms with pesticide resistance, specifically bacteriaand plants. These organisms and compositions derived from them aredesirable for agricultural purposes. The compositions of the inventionare also useful for generating altered or improved toxin proteins thathave pesticidal activity, or for detecting the presence of toxinproteins or nucleic acids in products or organisms.

The following embodiments are encompassed by the present invention:

1. A recombinant nucleic acid molecule comprising a nucleotide sequenceencoding an amino acid sequence having pesticidal activity, wherein saidnucleotide sequence is selected from the group consisting of:

-   -   a) the nucleotide sequence set forth in any of SEQ ID NO:1-47;    -   b) a nucleotide sequence that encodes a polypeptide comprising        the amino acid sequence of any of SEQ ID NO:50-96; and    -   c) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 95% sequence identity to        the amino acid sequence of any of SEQ ID NO:50-96.

2. The recombinant nucleic acid molecule of embodiment 1, wherein saidnucleotide sequence is a synthetic sequence that has been designed forexpression in a plant.

3. The recombinant nucleic acid molecule of embodiment 2, wherein saidsequence is set forth in any of SEQ ID NO:97-203.

4. The recombinant nucleic acid molecule of claim 1, wherein saidnucleotide sequence is operably linked to a promoter capable ofdirecting expression of said nucleotide sequence in a plant cell.

5. A vector comprising the nucleic acid molecule of embodiment 1.

6. The vector of embodiment 5, further comprising a nucleic acidmolecule encoding a heterologous polypeptide.

7. A host cell that contains the vector of embodiment 5.

8. The host cell of embodiment 7 that is a bacterial host cell. 9. Thehost cell of embodiment 7 that is a plant cell.

10. A transgenic plant comprising the host cell of embodiment 9.

11. The transgenic plant of embodiment 10, wherein said plant isselected from the group consisting of maize, sorghum, wheat, cabbage,sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean,sugarbeet, sugarcane, tobacco, barley, and oilseed rape.

12. A transgenic seed comprising the nucleic acid molecule of embodiment1.

13. A recombinant polypeptide with pesticidal activity, selected fromthe group consisting of:

-   -   a) a polypeptide comprising the amino acid sequence of any of        SEQ ID NO:50-96;    -   b) a polypeptide comprising an amino acid sequence having at        least 95% sequence identity to the amino acid sequence of any of        SEQ ID NO:50-96; and    -   c) a polypeptide that is encoded by any of SEQ ID NO:1-47.

14. The polypeptide of embodiment 13 further comprising heterologousamino acid sequences.

15. A composition comprising the recombinant polypeptide of embodiment13.

16. The composition of embodiment 15, wherein said composition isselected from the group consisting of a powder, dust, pellet, granule,spray, emulsion, colloid, and solution.

17. The composition of embodiment 15, wherein said composition isprepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof bacterial cells.

18. The composition of embodiment 15, comprising from about 1% to about99% by weight of said polypeptide. 19. A method for controlling alepidopteran, coleopteran, heteropteran, nematode, or dipteran pestpopulation comprising contacting said population with apesticidally-effective amount of the polypeptide of embodiment 13.

20. A method for killing a lepidopteran, coleopteran, heteropteran,nematode, or dipteran pest, comprising contacting said pest with, orfeeding to said pest, a pesticidally-effective amount of the polypeptideof embodiment 13.

21. A method for producing a polypeptide with pesticidal activity,comprising culturing the host cell of embodiment 7 under conditions inwhich the nucleic acid molecule encoding the polypeptide is expressed.

22. A plant having stably incorporated into its genome a DNA constructcomprising a nucleotide sequence that encodes a protein havingpesticidal activity, wherein said nucleotide sequence is selected fromthe group consisting of:

-   -   a) the nucleotide sequence set forth in any of SEQ ID NO:1-47;    -   b) a nucleotide sequence that encodes a polypeptide comprising        the amino acid sequence of any of SEQ ID NO:50-96; and    -   c) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 95% sequence identity to        the amino acid sequence of any of SEQ ID NO:50-96;        wherein said nucleotide sequence is operably linked to a        promoter that drives expression of a coding sequence in a plant        cell.

23. The plant of embodiment 22, wherein said plant is a plant cell.

24. A method for protecting a plant from a pest, comprising expressingin a plant or cell thereof a nucleotide sequence that encodes apesticidal polypeptide, wherein said nucleotide sequence is selectedfrom the group consisting of:

-   -   a) the nucleotide sequence set forth in any of SEQ ID NO:1-47;    -   b) a nucleotide sequence that encodes a polypeptide comprising        the amino acid sequence of any of SEQ ID NO:50-96; and    -   c) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 95% sequence identity to        the amino acid sequence of any of SEQ ID NO:50-96.

25. The method of embodiment 24, wherein said plant produces apesticidal polypeptide having pesticidal activity against alepidopteran, coleopteran, heteropteran, nematode, or dipteran pest.

26. A method for increasing yield in a plant comprising growing in afield a plant of or a seed thereof having stably incorporated into itsgenome a DNA construct comprising a nucleotide sequence that encodes aprotein having pesticidal activity, wherein said nucleotide sequence isselected from the group consisting of:

-   -   a) the nucleotide sequence set forth in any of SEQ ID NO:1-47;    -   b) a nucleotide sequence that encodes a polypeptide comprising        the amino acid sequence of any of SEQ ID NO:50-96; and    -   c) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 95% sequence identity to        the amino acid sequence of any of SEQ ID NO:50-96;        wherein said field is infested with a pest against which said        polypeptide has pesticidal activity.

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating pest resistance in organisms, particularly plants or plantcells. The methods involve transforming organisms with a nucleotidesequence encoding a toxin protein of the invention. In particular, thenucleotide sequences of the invention are useful for preparing plantsand microorganisms that possess pesticidal activity. Thus, transformedbacteria, plants, plant cells, plant tissues and seeds are provided.Compositions are toxin nucleic acids and proteins of Bacillusthuringiensis. The sequences find use in the construction of expressionvectors for subsequent transformation into organisms of interest, asprobes for the isolation of other toxin genes, and for the generation ofaltered pesticidal proteins by methods known in the art, such as domainswapping or DNA shuffling. The proteins find use in controlling orkilling lepidopteran, coleopteran, and nematode pest populations, andfor producing compositions with pesticidal activity.

By “toxin” is intended a sequence disclosed herein that has toxicactivity against one or more pests, including, but not limited to,members of the Lepidoptera, Diptera, and Coleoptera orders or members ofthe Nematoda phylum, or a protein that has homology to such a protein.In some cases, toxin proteins have been isolated from Bacillus sp. Inanother embodiment, the toxins have been isolated from other organisms,including Clostridium bifermentans and Paenibacillus popilliae. Toxinproteins include amino acid sequences deduced from the full-lengthnucleotide sequences disclosed herein, and amino acid sequences that areshorter than the full-length sequences, either due to the use of analternate downstream start site, or due to processing that produces ashorter protein having pesticidal activity. Processing may occur in theorganism the protein is expressed in, or in the pest after ingestion ofthe protein.

In various embodiments, the sequences disclosed herein have homology todelta-endotoxin proteins. Delta-endotoxins include proteins identifiedas cry1 through cry53, cyt1 and cyt2, and Cyt-like toxin. There arecurrently over 250 known species of delta-endotoxins with a wide rangeof specificities and toxicities. For an expansive list see Crickmore etal. (1998), Microbiol. Mol. Biol. Rev. 62:807-813, and for regularupdates see Crickmore et al. (2003) “Bacillus thuringiensis toxinnomenclature,” at www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index. Insome embodiments, the delta-endotoxin sequences disclosed herein includethe nucleotide sequences set forth in any of SEQ ID NO:1-47 or 97-203,the amino acid sequences set forth in any of SEQ ID NO:50-96, as well asvariants and fragments thereof.

Cry8 Homologues

In one embodiment, the sequences disclosed herein have homology to thecry8 family of delta-endotoxins. The cry8 family of delta-endotoxins hasbeen shown to be toxic to coleopteran insects. For example,coleopteran-active Cry8 mutants are described in U.S. Pat. No. 7,105,332(herein incorporated by reference in its entirety). Also presented inthe '332 patent is a cry8 homology model built from the solved structureof the Cry3A gene (Li et al. (1991) Nature 353:815 821) which providesinsight into the relationship between structure and function of theendotoxin

In some embodiments, the cry8 homologues encompassed herein include thenucleotide sequences include the nucleotide sequences set forth in SEQID NO:1, 2, and 3, as well as the amino acid sequences set forth in SEQID NO:50, 51, and 52. Biologically-active variants and fragments ofthese sequences are also encompassed.

Cry7-Like Sequences

In one embodiment, the sequences disclosed herein are cry7-likedelta-endotoxins. In various embodiments, the cry7-like sequences of theinvention include the nucleotide sequence set forth in SEQ ID NO:4, theamino acid sequence set forth in SEQ ID NO:53, as well asbiologically-active variants and fragments thereof.

cry1I Homolog

In another embodiment, the sequences disclosed herein have homology tothe cry1I family of delta-endotoxins. In some embodiments, the cry1Ihomologues include the nucleotide sequence set forth in SEQ ID NO:5, theamino acid sequence set forth in SEQ ID NO:54, and biologically-activevariants and fragments thereof.

The cry1I genes (formerly cryV genes), encode proteins of around 70 to81 kDa that do not accumulate in the crystal (Choi et al. (2000) Curr.Microbiol. 41:65-69; Gleave et al. (1993) Appl. Environ. Microbiol.59:1683-1687; Kostichka et al. (1996) J. Bacteriol. 178:2141-2144; U.S.Pat. Nos. 6,232,439; 5,723,758; Selvapandiyan et al. (2001) Appl.Environ. Microbiol. 67:5855-5858; Shin et al. (1995) Appl. Environ.Microbiol. 61:2402-2407; Song et al. (2003) Appl. Environ. Microbiol.69:5207-5211; Tailor et al. (1992) Mol. Microbiol. 6:1211-1217; andTounsi et al. (2003) J. Appl. Microbiol. 95:23-28); these have beenclassified as Cry1I proteins due to their similarity with those in theCry1 group (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev.62:807-813). The effectiveness of Cry1I in protecting transformed plantsfrom insect attack has been demonstrated (Lagnaoui et al. (2001) CIPProgram Rep. 1999-2000:117-121; Liu et al. (2004) Acta Biochim. Biophys.Sin. 36:309-313; and Selvapandiyan et al. (1998) Mol. Breed. 4:473-478).cry1I genes are usually either silent or expressed in the vegetativephase and secreted into the growth suspension (Kostichka et al. (1996)J. Bacteriol. 178:2141-2144; Selvapandiyan et al. (2001) Appl. Environ.Microbiol. 67:5855-5858; Song et al. (2003) Appl. Environ. Microbiol.69:5207-5211; and Tounsi et al. (2003) J. Appl. Microbiol. 95:23-28).Cry1I proteins have a broader host range than most other Cry1 proteins,and the hosts include important species of lepidopteran and coleopteranpests (Tailor et al. (1992) Mol. Microbiol. 6:1211-1217).

Cry9 Homologues

In another embodiment, the sequences disclosed herein have homology tothe cry9 family of delta-endotoxins. In some embodiments, the cry9homologues include the nucleotide sequence set forth in SEQ ID NO:6, theamino acid sequence set forth in SEQ ID NO:55, and biologically-activevariants and fragments thereof.

Cry4 Homologues

In another embodiment, the sequences disclosed herein have homology tothe cry4 family of delta-endotoxins. In various embodiments, the cry4homologues encompassed herein include the nucleotide sequences set forthin SEQ ID NO:7, 8, 9, 10, 11, and 12, the amino acid sequences set forthin SEQ ID NO:56, 57, 58, 59, 60, and 61, and biologically-activevariants and fragments thereof.

The cry4 family of delta-endotoxins has been shown to have activityagainst dipteran pests. Angsuthanasombat et al. ((2004) Journal ofBiochemistry and Molecular Biology 37(3):304-313, which is hereinincorporated by reference in its entirety, particularly with respect tothe structural analysis of cry4) describe the 3-D structure of cry4 andcorrelate the structure with dipteran activity.

cryC35/53-Like Sequences

In another embodiment, the sequences disclosed herein arecryC35/cryC53-like sequences. In some embodiments, thecryC35/cryC53-like sequences include the nucleotide sequences set forthin SEQ ID NO:13, 14, 15, or 16, the amino acid sequences set forth inSEQ ID NO:62, 63, 64, and 65, and biologically-active variants andfragments thereof.

Cry21/Cry12-Like Sequences

In another embodiment, the sequences disclosed herein arecry21/cry12-like sequences. The cry12/21 family has been shown to haveactivity against nematode pests (Wei (2003) Proc.Natl.Acad.Sci. USA100(5):2760-2765 and European Patent No. 0462721A2, each of which isherein incorporated by reference). In various embodiments, thecry21/cry12-like sequences encompassed herein include the nucleotidesequences set forth in SEQ ID NO:17, 18, 19, and 20, the amino acidsequences set forth in SEQ ID NO:66, 67, 68, and 69, andbiologically-active variants and fragments thereof.

VIP-Like or Binary-Like Sequences

In another embodiment, the sequences disclosed herein are VIP-like orbinary-like proteins. Vegetative insecticidal proteins (VIPs) areinsecticidal proteins produced during vegetative growth of the bacteriaand are thus classified as exotoxins. The VIP gene shows insecticidalactivity against a variety of lepidopterans.

The VIP1/VIP2 toxins (see, for example, U.S. Pat. No. 5,770,696, hereinincorporated by reference in its entirety) are binary pesticidal toxinsthat exhibit strong activity on insects by a mechanism believed toinvolve receptor-mediated endocytosis followed by cellular toxification,similar to the mode of action of other binary (“A/B”) toxins. A/B toxinssuch as VIP, C2, CDT, CST, or the B. anthracis edema and lethal toxinsinitially interact with target cells via a specific, receptor-mediatedbinding of “B” components as monomers. These monomers then formhomoheptamers. The “B” heptamer-receptor complex then acts as a dockingplatform that subsequently binds and allows the translocation of anenzymatic “A” component(s) into the cytosol via receptor-mediatedendocytosis. Once inside the cell's cytosol, “A” components inhibitnormal cell function by, for example, ADP-ribosylation of G-actin, orincreasing intracellular levels of cyclic AMP (cAMP). See Barth et al.(2004) Microbiol Mol Biol Rev 68:373-402, herein incorporated byreference in its entirety.

Aside from the A/B type binary toxins, other types of binary toxins thatact as pesticidal proteins are known in the art. Cry34Ab 1 and Cry35Ab 1comprise a binary toxin with pesticidal activity that was identifiedfrom strain PS149B1 (Ellis et al. (2002) Appl Environ Microbiol.68:1137-45, herein incorporated by reference in its entirety). Thesetoxins have molecular masses of approximately 14 and 44 kDa,respectively. Other binary toxins with similar organization and homologyto Cry34Aa and Cry34Ab have been identified (Baum et al. (2004) ApplEnviron Microbiol. 70:4889-98, herein incorporated by reference in itsentirety).

BinA and BinB are proteins from Bacillus sphaericus that comprise amosquitocidal binary toxin protein (Baumann et al. (1991) Micriobiol.Rev. 55:425-36). Cry35 exhibits amino acid similarity to these BinA andBinB proteins. Cry36 (ET69) and Cry38 (ET75) (International PatentApplication No. WO/00/66742-B, herein incorporated by reference in itsentirety) are independently isolated peptides that also exhibit aminoacid similarity to BinA and BinB, and thus are likely to comprise binarytoxins.

In various embodiments, VIP-like or binary-like sequences encompassedherein include the nucleotide sequences set forth in SEQ ID NO:21, 22,23, and 24, the amino acid sequences set forth in SEQ ID NO:70, 71, 72,and 73, and biologically-active variants and fragments thereof.

MTX-Like Sequences

In yet another embodiment, the sequences encompassed herein are MTX-likesequences. The term “MTX” is used in the art to delineate a set ofpesticidal proteins that are produced by Bacillus sphaericus. The firstof these, often referred to in the art as MTX1, is synthesized as aparasporal crystal which is toxic to mosquitoes. The major components ofthe crystal are two proteins of 51 and 42 kDa, Since the presence ofboth proteins are required for toxicity, MTX1 is considered a “binary”toxin (Baumann et al. (1991) Microbiol. Rev. 55:425-436).

By analysis of different Bacillus sphaericus strains with differingtoxicities, two new classes of MTX toxins have been identified. MTX2 andMTX3 represent separate, related classes of pesticidal toxins thatexhibit pesticidal activity. See, for example, Baumann et al. (1991)Microbiol. Rev. 55:425-436, herein incorporated by reference in itsentirety. MTX2 is a 100-kDa toxin. More recently MTX3 has beenidentified as a separate toxin, though the amino acid sequence of MTX3from B. sphaericus is 38% identical to the MTX2 toxin of B. sphaericusSSII-1 (Liu, et al. (1996) Appl. Environ. Microbiol. 62: 2174-2176). Mtxtoxins may be useful for both increasing the insecticidal activity of B.sphaericus strains and managing the evolution of resistance to the Bintoxins in mosquito populations (Wirth et al. (2007) Appl EnvironMicrobiol 73(19):6066-6071).

In various embodiments, the MTX-like sequences include the nucleotidesequences set forth in SEQ ID NO:25, 26, 27, 28, and 29, the amino acidsequences set forth in SEQ ID NO:74, 75, 76, 77, and 78, andbiologically-active variants and fragments thereof.

Non-Cry Toxin

The present invention further comprises toxin sequences that are notdelta-endotoxins, but have been isolated from Bacillus. In oneembodiment, these toxins have homology to phosphatidylinositolphosphodiesterases (also referred to as phosphatidylinositol-specificphospholipase C (PI-PLC)). Phosphatidylinositol phosphodiesterasecleaves glycosylphosphatidylinositol (GPI) and phosphatidylinositol (PI)anchors. These proteins contain a PI-PLC X-box domain, and have beenisolated from cultures of Bacillus cereus, Bacillus thuringiensis,Staphylococcus aureus, and Clostridium novyi, which secrete the enzymeacross the bacterial membrane into the culture medium. The role of aminoacid residues located in the active site pocket ofphosphatidylinositol-specific phospholipase C (PI-PLC) from Bacilluscereus (Heinz et al. (1995) EMBO J. 14, 3855-3863, herein incorporatedby reference in its entirety) was investigated by site-directedmutagenesis, kinetics, and crystal structure analysis (Gassler et al.(1997) Biochemistry 36:12802-12813, herein incorporated by reference inits entirety).

In various embodiments, the toxin sequences disclosed herein include thenucleotide sequences set forth in SEQ ID NO:30, the amino acid sequenceset forth in SEQ ID NO:79, and biologically-active variants andfragments thereof.

Thus, provided herein are families of novel isolated nucleotidesequences that confer pesticidal activity. Also provided are the aminoacid sequences of the toxin proteins. The protein resulting fromtranslation of this gene allows cells to control or kill pests thatingest it.

Cry55 Homologs

In another embodiment, the sequences disclosed herein are cry55homologs. In some embodiments, the cry55 homologs include the nucleotidesequences set forth in SEQ ID NO:43 and 44, the amino acid sequences setforth in SEQ ID NO:92 and 93, and biologically-active variants andfragments thereof.

cry15A Homologs

In another embodiment, the sequences disclosed herein are cry15Ahomologs. Cry15A toxins have been shown to have activity againstlepidopteran pests (Rang et al. (2000) Curr Microbiol. 40(3):200-4). Insome embodiments, the cry15A homologs include the nucleotide sequencesset forth in SEQ ID NO:45, 46, and 47, the amino acid sequences setforth in SEQ ID NO:94, 95, and 96, and biologically-active variants andfragments thereof.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding toxin proteinsand polypeptides or biologically active portions thereof, as well asnucleic acid molecules sufficient for use as hybridization probes toidentify toxin encoding nucleic acids. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., recombinantDNA, cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs ofthe DNA or RNA generated using nucleotide analogs. The nucleic acidmolecule can be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolatedtoxin encoding nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. A toxin protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) of non-toxinprotein (also referred to herein as a “contaminating protein”).

Nucleotide sequences encoding the proteins of the present inventioninclude the sequence set forth in SEQ ID NO:1-47, and variants,fragments, and complements thereof. By “complement” is intended anucleotide sequence that is sufficiently complementary to a givennucleotide sequence such that it can hybridize to the given nucleotidesequence to thereby form a stable duplex. The corresponding amino acidsequence for the toxin protein encoded by this nucleotide sequence areset forth in SEQ ID NO:50-96.

Nucleic acid molecules that are fragments of these toxin encodingnucleotide sequences are also encompassed by the present invention. By“fragment” is intended a portion of the nucleotide sequence encoding atoxin protein. A fragment of a nucleotide sequence may encode abiologically active portion of a toxin protein, or it may be a fragmentthat can be used as a hybridization probe or PCR primer using methodsdisclosed below. Nucleic acid molecules that are fragments of a toxinnucleotide sequence comprise at least about 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550,2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150,3200, 3250, 3300, 3350 contiguous nucleotides, or up to the number ofnucleotides present in a full-length toxin encoding nucleotide sequencedisclosed herein depending upon the intended use. By “contiguous”nucleotides is intended nucleotide residues that are immediatelyadjacent to one another. Fragments of the nucleotide sequences of thepresent invention will encode protein fragments that retain thebiological activity of the toxin protein and, hence, retain pesticidalactivity. By “retains activity” is intended that the fragment will haveat least about 30%, at least about 50%, at least about 70%, 80%, 90%,95% or higher of the pesticidal activity of the toxin protein. Methodsfor measuring pesticidal activity are well known in the art. See, forexample, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrewset al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. ofEconomic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all ofwhich are herein incorporated by reference in their entirety.

A fragment of a toxin encoding nucleotide sequence that encodes abiologically active portion of a protein of the invention will encode atleast about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100 contiguous amino acids, or up to the total number of amino acidspresent in a full-length toxin protein of the invention. In someembodiments, the fragment is a proteolytic cleavage fragment. Forexample, the proteolytic cleavage fragment may have an N-terminal or aC-terminal truncation of at least about 100 amino acids, about 120,about 130, about 140, about 150, or about 160 amino acids relative toSEQ ID NO:50-96. In some embodiments, the fragments encompassed hereinresult from the removal of the C-terminal crystallization domain, e.g.,by proteolysis or by insertion of a stop codon in the coding sequence.

Preferred toxin proteins of the present invention are encoded by anucleotide sequence sufficiently identical to the nucleotide sequence ofSEQ ID NO:1-47. By “sufficiently identical” is intended an amino acid ornucleotide sequence that has at least about 60% or 65% sequenceidentity, about 70% or 75% sequence identity, about 80% or 85% sequenceidentity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of one ofSEQ ID NO:1-47, or across the entirety of one of SEQ ID NO:50-96). Thepercent identity between two sequences can be determined usingtechniques similar to those described below, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous totoxin-like nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to toxin proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST (in BLAST 2.0) can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively,PSI-Blast can be used to perform an iterated search that detects distantrelationships between molecules. See Altschul et al. (1997) supra. Whenutilizing BLAST, Gapped BLAST, and PSI-Blast programs, the defaultparameters of the respective programs (e.g., BLASTX and BLASTN) can beused. Alignment may also be performed manually by inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The invention also encompasses variant nucleic acid molecules.“Variants” of the toxin encoding nucleotide sequences include thosesequences that encode the toxin proteins disclosed herein but thatdiffer conservatively because of the degeneracy of the genetic code aswell as those that are sufficiently identical as discussed above.Naturally occurring allelic variants can be identified with the use ofwell-known molecular biology techniques, such as polymerase chainreaction (PCR) and hybridization techniques as outlined below. Variantnucleotide sequences also include synthetically derived nucleotidesequences that have been generated, for example, by using site-directedmutagenesis but which still encode the toxin proteins disclosed in thepresent invention as discussed below. Variant proteins encompassed bythe present invention are biologically active, that is they continue topossess the desired biological activity of the native protein, that is,retaining pesticidal activity. By “retains activity” is intended thatthe variant will have at least about 30%, at least about 50%, at leastabout 70%, or at least about 80% of the pesticidal activity of thenative protein. Methods for measuring pesticidal activity are well knownin the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83: 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone etal. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedtoxin proteins, without altering the biological activity of theproteins. Thus, variant isolated nucleic acid molecules can be createdby introducing one or more nucleotide substitutions, additions, ordeletions into the corresponding nucleotide sequence disclosed herein,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, conservative amino acid substitutions may be made at one ormore predicted, nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from the wild-typesequence of a toxin protein without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of the amino acidsequences of the present invention and known toxin sequences. Examplesof residues that are conserved but that may allow conservative aminoacid substitutions and still retain activity include, for example,residues that have only conservative substitutions between all proteinscontained in an alignment of the amino acid sequences of the presentinvention and known toxin sequences. However, one of skill in the artwould understand that functional variants may have minor conserved ornonconserved alterations in the conserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer toxin activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingtoxin sequences can be identified, such sequences having substantialidentity to the sequences of the invention. See, for example, Sambrookand Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, NY).

In a hybridization method, all or part of the toxin nucleotide sequencecan be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, 2001, supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known toxin-encoding nucleotidesequence disclosed herein. Degenerate primers designed on the basis ofconserved nucleotides or amino acid residues in the nucleotide sequenceor encoded amino acid sequence can additionally be used. The probetypically comprises a region of nucleotide sequence that hybridizesunder stringent conditions to at least about 12, at least about 25, atleast about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400consecutive nucleotides of toxin encoding nucleotide sequence of theinvention or a fragment or variant thereof. Methods for the preparationof probes for hybridization are generally known in the art and aredisclosed in Sambrook and Russell, 2001, supra herein incorporated byreference.

For example, an entire toxin sequence disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding toxin-like sequences and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, or at least about 20 nucleotides inlength. Such probes may be used to amplify corresponding toxin sequencesfrom a chosen organism by PCR. This technique may be used to isolateadditional coding sequences from a desired organism or as a diagnosticassay to determine the presence of coding sequences in an organism.Hybridization techniques include hybridization screening of plated DNAlibraries (either plaques or colonies; see, for example, Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions.

By “stringent conditions” or “stringent hybridization conditions” isintended conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≥90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Toxin proteins are also encompassed within the present invention. By“toxin protein” is intended a protein having the amino acid sequence setforth in SEQ ID NO:50-96. Fragments, biologically active portions, andvariants thereof are also provided, and may be used to practice themethods of the present invention.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in any of SEQ ID NO:50-96 and that exhibitpesticidal activity. A biologically active portion of a toxin proteincan be a polypeptide that is, for example, 10, 25, 50, 100 or more aminoacids in length. Such biologically active portions can be prepared byrecombinant techniques and evaluated for pesticidal activity. Methodsfor measuring pesticidal activity are well known in the art. See, forexample, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrewset al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. ofEconomic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all ofwhich are herein incorporated by reference in their entirety. As usedhere, a fragment comprises at least 8 contiguous amino acids of SEQ IDNO:50-96. The invention encompasses other fragments, however, such asany fragment in the protein greater than about 10, 20, 30, 50, 100, 150,200, 250, 300, 350, 400, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of any of SEQ ID NO:50-96. Variants alsoinclude polypeptides encoded by a nucleic acid molecule that hybridizesto the nucleic acid molecule of SEQ ID NO:1-47, or a complement thereof,under stringent conditions. Variants include polypeptides that differ inamino acid sequence due to mutagenesis. Variant proteins encompassed bythe present invention are biologically active, that is they continue topossess the desired biological activity of the native protein, that is,retaining pesticidal activity. Methods for measuring pesticidal activityare well known in the art. See, for example, Czapla and Lang (1990) J.Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293;and U.S. Pat. No. 5,743,477, all of which are herein incorporated byreference in their entirety.

Bacterial genes, such as the axmi genes of this invention, quite oftenpossess multiple methionine initiation codons in proximity to the startof the open reading frame. Often, translation initiation at one or moreof these start codons will lead to generation of a functional protein.These start codons can include ATG codons. However, bacteria such asBacillus sp. also recognize the codon GTG as a start codon, and proteinsthat initiate translation at GTG codons contain a methionine at thefirst amino acid. Furthermore, it is not often determined a priori whichof these codons are used naturally in the bacterium. Thus, it isunderstood that use of one of the alternate methionine codons may alsolead to generation of toxin proteins that encode pesticidal activity.These toxin proteins are encompassed in the present invention and may beused in the methods of the present invention.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that DNA sequences of a toxin may be altered by variousmethods, and that these alterations may result in DNA sequences encodingproteins with amino acid sequences different than that encoded by atoxin of the present invention. This protein may be altered in variousways including amino acid substitutions, deletions, truncations, andinsertions of one or more amino acids of SEQ ID NO:50-96, including upto about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 45, about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 100, about 105, about 110, about115, about 120, about 125, about 130 or more amino acid substitutions,deletions or insertions.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a toxin protein can be preparedby mutations in the DNA. This may also be accomplished by one of severalforms of mutagenesis and/or in directed evolution. In some aspects, thechanges encoded in the amino acid sequence will not substantially affectthe function of the protein. Such variants will possess the desiredpesticidal activity. However, it is understood that the ability of atoxin to confer pesticidal activity may be improved by the use of suchtechniques upon the compositions of this invention. For example, one mayexpress a toxin in host cells that exhibit high rates of basemisincorporation during DNA replication, such as XL-1 Red (Stratagene).After propagation in such strains, one can isolate the toxin DNA (forexample by preparing plasmid DNA, or by amplifying by PCR and cloningthe resulting PCR fragment into a vector), culture the toxin mutationsin a non-mutagenic strain, and identify mutated toxin genes withpesticidal activity, for example by performing an assay to test forpesticidal activity. Generally, the protein is mixed and used in feedingassays. See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests. Examples of mutations that result in increasedtoxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev.62:775-806.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent toxin protein coding regions can be used to create a new toxinprotein possessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a toxin gene ofthe invention and other known toxin genes to obtain a new gene codingfor a protein with an improved property of interest, such as anincreased insecticidal activity. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating altereddelta-endotoxin proteins. Domains II and III may be swapped betweendelta-endotoxin proteins, resulting in hybrid or chimeric toxins withimproved pesticidal activity or target spectrum. Methods for generatingrecombinant proteins and testing them for pesticidal activity are wellknown in the art (see, for example, Naimov et al. (2001) Appl. Environ.Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ.Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem.266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930;Rang et al. 91999) Appl. Environ. Microbiol. 65:2918-2925).

Vectors

A toxin sequence of the invention may be provided in an expressioncassette for expression in a plant of interest. By “plant expressioncassette” is intended a DNA construct that is capable of resulting inthe expression of a protein from an open reading frame in a plant cell.Typically these contain a promoter and a coding sequence. Often, suchconstructs will also contain a 3′ untranslated region. Such constructsmay contain a “signal sequence” or “leader sequence” to facilitateco-translational or post-translational transport of the peptide tocertain intracellular structures such as the chloroplast (or otherplastid), endoplasmic reticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.By “leader sequence” is intended any sequence that when translated,results in an amino acid sequence sufficient to trigger co-translationaltransport of the peptide chain to a sub-cellular organelle. Thus, thisincludes leader sequences targeting transport and/or glycosylation bypassage into the endoplasmic reticulum, passage to vacuoles, plastidsincluding chloroplasts, mitochondria, and the like.

By “plant transformation vector” is intended a DNA molecule that isnecessary for efficient transformation of a plant cell. Such a moleculemay consist of one or more plant expression cassettes, and may beorganized into more than one “vector” DNA molecule. For example, binaryvectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a nucleic acid constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

“Promoter” refers to a nucleic acid sequence that functions to directtranscription of a downstream coding sequence. The promoter togetherwith other transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) are necessary for theexpression of a DNA sequence of interest.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the toxin sequence to be under thetranscriptional regulation of the regulatory regions.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and a translationaland transcriptional termination region (i.e., termination region)functional in plants. The promoter may be native or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequence ofthe invention. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

In one embodiment, the toxin is targeted to the chloroplast forexpression. In this manner, where the toxin is not directly insertedinto the chloroplast, the expression cassette will additionally containa nucleic acid encoding a transit peptide to direct the toxin to thechloroplasts. Such transit peptides are known in the art. See, forexample, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clarket al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987)Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res.Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

The toxin gene to be targeted to the chloroplast may be optimized forexpression in the chloroplast to account for differences in codon usagebetween the plant nucleus and this organelle. In this manner, thenucleic acids of interest may be synthesized using chloroplast-preferredcodons. See, for example, U.S. Pat. No. 5,380,831, herein incorporatedby reference.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated exogenous nucleic acid sequences or DNA fragments into theplant cell. These nucleic acid sequences include those that areexogenous, or not present in the untransformed plant cell, as well asthose that may be endogenous, or present in the untransformed plantcell.

“Heterologous” generally refers to the nucleic acid sequences that arenot endogenous to the cell or part of the native genome in which theyare present, and have been added to the cell by infection, transfection,microinjection, electroporation, microprojection, or the like.

The transgenic plants of the invention express one or more of thepesticidal sequences disclosed herein. In various embodiments, thetransgenic plant further comprises one or more additional genes forinsect resistance, for example, one or more additional genes forcontrolling coleopteran, lepidopteran, heteropteran, or nematode pests.It will be understood by one of skill in the art that the transgenicplant may comprise any gene imparting an agronomic trait of interest.

Transformation of plant cells can be accomplished by one of severaltechniques known in the art. The toxin gene of the invention may bemodified to obtain or enhance expression in plant cells. Typically aconstruct that expresses such a protein would contain a promoter todrive transcription of the gene, as well as a 3′ untranslated region toallow transcription termination and polyadenylation. The organization ofsuch constructs is well known in the art. In some instances, it may beuseful to engineer the gene such that the resulting peptide is secreted,or otherwise targeted within the plant cell. For example, the gene canbe engineered to contain a signal peptide to facilitate transfer of thepeptide to the endoplasmic reticulum. It may also be preferable toengineer the plant expression cassette to contain an intron, such thatmRNA processing of the intron is required for expression.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector”. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas “binary vectors”. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “gene of interest” (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the toxin are located between the leftand right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Generation oftransgenic plants may be performed by one of several methods, including,but not limited to, microinjection, electroporation, direct genetransfer, introduction of heterologous DNA by Agrobacterium into plantcells (Agrobacterium-mediated transformation), bombardment of plantcells with heterologous foreign DNA adhered to particles, ballisticparticle acceleration, aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066),Lec1 transformation, and various other non-particle direct-mediatedmethods to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the toxin is then tested by hybridizing the filter to aradioactive probe derived from a toxin, by methods known in the art(Sambrook and Russell, 2001, supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thetoxin gene by standard procedures (Sambrook and Russell, 2001, supra)using antibodies that bind to one or more epitopes present on the toxinprotein.

Pesticidal Activity in Plants

In another aspect of the invention, one may generate transgenic plantsexpressing a toxin that has pesticidal activity. Methods described aboveby way of example may be utilized to generate transgenic plants, but themanner in which the transgenic plant cells are generated is not criticalto this invention. Methods known or described in the art such asAgrobacterium-mediated transformation, biolistic transformation, andnon-particle-mediated methods may be used at the discretion of theexperimenter. Plants expressing a toxin may be isolated by commonmethods described in the art, for example by transformation of callus,selection of transformed callus, and regeneration of fertile plants fromsuch transgenic callus. In such process, one may use any gene as aselectable marker so long as its expression in plant cells confersability to identify or select for transformed cells.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes that encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, genesthat provide resistance to plant herbicides such as glyphosate,bromoxynil, or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990)Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).Additionally, the genes disclosed herein are useful as markers to assesstransformation of bacterial or plant cells. Methods for detecting thepresence of a transgene in a plant, plant organ (e.g., leaves, stems,roots, etc.), seed, plant cell, propagule, embryo or progeny of the sameare well known in the art. In one embodiment, the presence of thetransgene is detected by testing for pesticidal activity.

Fertile plants expressing a toxin may be tested for pesticidal activity,and the plants showing optimal activity selected for further breeding.Methods are available in the art to assay for pest activity. Generally,the protein is mixed and used in feeding assays. See, for exampleMarrone et al. (1985) J. of Economic Entomology 78:290-293.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

Use in Pest Control

General methods for employing strains comprising a nucleotide sequenceof the present invention, or a variant thereof, in pesticide control orin engineering other organisms as pesticidal agents are known in theart. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains containing a nucleotide sequence of the presentinvention, or a variant thereof, or the microorganisms that have beengenetically altered to contain a pesticidal gene and protein may be usedfor protecting agricultural crops and products from pests. In one aspectof the invention, whole, i.e., unlysed, cells of a toxin(pesticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target pest(s).

Alternatively, the pesticide is produced by introducing a toxin geneinto a cellular host. Expression of the toxin gene results, directly orindirectly, in the intracellular production and maintenance of thepesticide. In one aspect of this invention, these cells are then treatedunder conditions that prolong the activity of the toxin produced in thecell when the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated pesticides may then be formulated in accordance withconventional techniques for application to the environment hosting atarget pest, e.g., soil, water, and foliage of plants. See, for exampleEPA 0192319, and the references cited therein. Alternatively, one mayformulate the cells expressing a gene of this invention such as to allowapplication of the resulting material as a pesticide.

Pesticidal Compositions

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be fertilizers, weed killers, cryoprotectants,surfactants, detergents, pesticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, dipteran, coleopteran, or nematode pests may be killed orreduced in numbers in a given area by the methods of the invention, ormay be prophylactically applied to an environmental area to preventinfestation by a susceptible pest. Preferably the pest ingests, or iscontacted with, a pesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations may also vary with respect to climatic conditions,environmental considerations, and/or frequency of application and/orseverity of pest infestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference.

Alternatively, the toxin genes could be cloned in Pseudomonas spp., thusexpressing the proteins and microencapsulating them in the bacterialcell wall. Microencapsulated toxin could be used in spray applicationsalone or in rotations with B. thuringiensis-based insecticidescontaining other toxins.

The plants can also be treated with one or more chemical compositions,including one or more herbicide, insecticides, or fungicides. Exemplarychemical compositions include: Fruits/Vegetables Herbicides: Atrazine,Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine,Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat,Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam;Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuriengiensis,Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin,Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin,Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate,Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran,Fluacrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen,Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr,Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide,Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen,Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb,Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Forthiazate,Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox,Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron,Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam,Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim,Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph,Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid,Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione,Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron,Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet,Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin,Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos,Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron,Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin,Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos,Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin,Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan,Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides:Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron,Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron,Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet,Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid,Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor,Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; RiceInsecticides: Diazinon, Fenitrothion, Fenobucarb, Monocrotophos,Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid,Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran,Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin,Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos,Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran, Organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga.Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea,while suborder Polyphaga includes the superfamilies Hydrophiloidea,Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea,Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea,Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea,Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes thefamilies Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoideaincludes the family Gyrinidae. Superfamily Hydrophiloidea includes thefamily Hydrophilidae. Superfamily Staphylinoidea includes the familiesSilphidae and Staphylinidae. Superfamily Cantharoidea includes thefamilies Cantharidae and Lampyridae. Superfamily Cleroidea includes thefamilies Cleridae and Dermestidae. Superfamily Elateroidea includes thefamilies Elateridae and Buprestidae. Superfamily Cucujoidea includes thefamily Coccinellidae. Superfamily Meloidea includes the family Meloidae.Superfamily Tenebrionoidea includes the family Tenebrionidae.Superfamily Scarabaeoidea includes the families Passalidae andScarabaeidae. Superfamily Cerambycoidea includes the familyCerambycidae. Superfamily Chrysomeloidea includes the familyChrysomelidae. Superfamily Curculionoidea includes the familiesCurculionidae and Scolytidae.

The order Diptera includes the Suborders Nematocera, Brachycera, andCyclorrhapha. Suborder Nematocera includes the families Tipulidae,Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae,Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the familiesStratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae,and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschizaand Aschiza. Division Aschiza includes the families Phoridae, Syrphidae,and Conopidae. Division Aschiza includes the Sections Acalyptratae andCalyptratae. Section Acalyptratae includes the families Otitidae,Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptrataeincludes the families Hippoboscidae, Oestridae, Tachinidae,Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae,Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae,Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae,and Tineidae.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Insect pests of the invention for the major crops include: Maize:Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm;Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which saidpolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a lepidopteran, coleopteran,dipteran, hemipteran, or nematode pest, and said field is infested witha lepidopteran, hemipteran, coleopteran, dipteran, or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. By “biomass” is intended anymeasured plant product. An increase in biomass production is anyimprovement in the yield of the measured plant product. Increasing plantyield has several commercial applications. For example, increasing plantleaf biomass may increase the yield of leafy vegetables for human oranimal consumption. Additionally, increasing leaf biomass can be used toincrease production of plant-derived pharmaceutical or industrialproducts. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing a pesticidal protein disclosedherein. Expression of the pesticidal protein results in a reducedability of a pest to infest or feed on the plant, thus improving plantyield.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1. Identification of Novel Genes

Novel pesticidal genes are identified from the bacterial strainsdescribed herein using methods such as:

Method 1

-   -   Preparation of extrachromosomal DNA from the strain, which        includes plasmids that typically harbor delta-endotoxin genes    -   Mechanical shearing of extrachromosomal DNA to generate        size-distributed fragments    -   Cloning of ˜2 Kb to ˜10 Kb fragments of extrachromosomal DNA    -   Outgrowth of ˜1500 clones of the extrachromosomal DNA    -   Partial sequencing of the 1500 clones using primers specific to        the cloning vector (end reads)    -   Identification of putative toxin genes via homology analysis via        the MiDAS approach (as described in U.S. Patent Publication No.        20040014091, which is herein incorporated by reference in its        entirety)    -   Sequence finishing (walking) of clones containing fragments of        the putative toxin genes of interest

Method 2

-   -   Preparation of extrachromosomal DNA from the strain (which        contains a mixture of some or all of the following: plasmids of        various size; phage chromosomes; genomic DNA fragments not        separated by the purification protocol; other uncharacterized        extrachromosomal molecules)    -   Mechanical or enzymatic shearing of the extrachromosomal DNA to        generate size-distributed fragments    -   Sequencing of the fragmented DNA by high-throughput        pyrosequencing methods    -   Identification of putative toxin genes via homology and/or other        computational analyses    -   Sequence finishing of the gene of interest by one of several PCR        or cloning strategies (e.g. TAIL-PCR).

Example 2. Discovery of Novel Pesticidal Genes from Bacillusthuringiensis Having Homology to Cry8

TABLE 1 cry8 homologues Amino Molecular acid Gene weight ClosestNucleotide SEQ name Strain (kD) homolog SEQ ID NO ID NO Axmi128 ATX13034137.6 49% 1 50 Cry8Bb1 Axmi141 ATX12996 139.1 50% 2 51 Cry8Bc1 Axmi146ATX12996 136.5 46% 3 52 Cry8Bb1

Example 3. Discovery of Novel Cry7-Like Pesticidal Genes from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 2 using the methods disclosed in Example 1.

TABLE 2 cry7-like sequences Amino Molecular acid Gene weight ClosestNucleotide SEQ name Strain (kD) homolog SEQ ID NO ID NO Axmi152 ATX13006128.4 67% 4 53 Cry7Aa1

Example 4. Discovery of a Novel cry1I Homolog from Bacillusthuringiensis

A novel pesticidal gene was identified from the bacterial strain listedin Table 3 using the methods disclosed in Example 1.

TABLE 3 cry 11 homolog Molecular Amino Gene weight Closest Nucleotideacid SEQ name Strain (kD) homolog SEQ ID NO ID NO Axmi156 ATX14775 84.895% Cry1Ie1 5 54

Example 5. Discovery of Novel Cry9 Homologues from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 4 using the methods disclosed in Example 1.

TABLE 4 cry9 homologues Molecular Nucleo- Amino Gene weight Closest tideSEQ acid SEQ name Strain (kD) homolog ID NO ID NO Axmi162 ATX14775 131.690% Cry9Db1 6 55

Example 6. Discovery of Novel Cry4 Homologues from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 5 using the methods disclosed in Example 1.

TABLE 5 cry4 homologues Molecular Nucleo- Amino Gene weight Closest tideSEQ acid SEQ name Strain (kD) homolog ID NO ID NO Axmi131 ATX13029 78.958% Cry4Ba1 7 56 Axmi139 ATX13027 68.5 38% Cry4Ba1 8 57 57% Axmi081Axmi144 ATX15076 71.7 34% Cry29Aa1 9 58 Axmi145 ATX15076 64.6 64%Cry4Aa1 10 59 Axmi167 ATX15076 139.7 46% Cry4Aa2 11 60 Axmi140 ATX13027161.9 31% Cry4Ba2 12 61 61% Axmi075

Example 7. Discovery of Novel cryC53/cryC53-Like Sequences from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 6 using the methods disclosed in Example 1. The full lengthsequence encoding both axmi-153 and axmi-154 is set forth in SEQ IDNO:48. The full length sequence encoding both axmi-157 and axmi-158 isset forth in SEQ ID NO:49.

TABLE 6 cryC35/53-like sequences Molecular Nucleo- Amino Gene weightClosest tide SEQ acid SEQ name Strain (kD) homolog ID NO ID NO Axmi1531ATX13037 37.5 54% CryC35 13 62 94% Axmi063 Axmi1542 ATX13037 39.3 44%CryC53 14 63 76% Axmi064 Axmi1573 ATX13049 37.2 38% CryC35 15 64 46%Axmi033 Axmi1584 ATX13049 36.2 31% CryC53 16 65 40% Axmi064 ¹pairs withAxmi154, ²pairs with Axmi153, ³pairs with Axmi158, ⁴pairs with Axmi157

Example 8. Discovery of Novel Cry21/Cry12-Like Sequences from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 7 using the methods disclosed in Example 1.

TABLE 7 cry21/cry12-like sequences Molecular Nucleo Amino Gene weightClosest tide SEQ acid SEQ name Strain (kD) homolog ID NO ID NO Axmi155ATX13020 146.4 37% Cry21Ba1 17 66 Axmi169 ATX13053 146.4 45% Cry21Ba1 1867 Axmi170 ATX13053 146.4 52% Cry21Ba1 19 68 Axmi171 ATX24692 95.2 22%Cry12Aa2 204 205

Example 9. Discovery of Novel VIP-Like or Binary-Like Sequences fromBacillus thuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 8 using the methods disclosed in Example 1.

TABLE 8 VIP-like or binary-like sequences Molecular Nucleo- Amino Geneweight Closest tide SEQ acid SEQ name Strain (kD) homolog ID NO ID NOAxmi135 ATX13015 98.0 71% Vip1Bb1 21 70 Axmi136 ATX13015 55.6 78%Vip2Ad1 22 71 Axmi142 ATX13038 41.4 24% BinA 23 72 Axmi149 ATX13059100.1 32% CdtB 24 73

Example 10. Discovery of Novel MTX-Like Sequences from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 9 using the methods disclosed in Example 1.

TABLE 9 MTX-like sequences Molecular Nucleo- Amino Gene weight Closesttide SEQ acid SEQ name Strain (kD) homolog ID NO ID NO Axmi130 ATX1302532.9 21% Mtx3 25 74 Axmi177 ATX25337 37.0 14% Mtx2 26 75 21% Axmi019Axmi178 ATX25743 36.4 17% Mtx2 27 76 Axmi179 ATX25743 36.1 18% Mtx2 2877 22% Axmi034 Axmi180 ATX26054 35.6 22% Mtx2 29 78 35% Axmi122

Example 11. Discovery of a Novel Toxin Sequence from Bacillusthuringiensis

A novel pesticidal gene was identified from the bacterial strain listedin Table 10 using the methods disclosed in Example 1.

TABLE 10 Molecular Nucleo- Amino Gene weight Closest tide SEQ acid SEQname Strain (kD) homolog ID NO ID NO Axmi133 ATX13029 57.9 42% 1- 30 79phosphati- dylinositol phospho- diesterase

Example 12. Discovery of Novel Toxin Sequences from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 11 using the methods disclosed in Example 1.

TABLE 11 Cry homologues Molecular Nucleo- Amino Gene weight Closest tideSEQ acid SEQ name Strain (kD) homolog ID NO ID NO Axmi143 ATX13053 77.130% Cry28Aa2 31 80 Axmi165 ATX25337 144.9 54% Cry32Aa1 32 81 Axmi166ATX25204 141.4 55% Cry32Aa1 33 82 Axmi168 ATX15076 77.5 38% Cry10Aa1 3483 50% Axmi125 Axmi172 ATX24692 84.7 31% Cry2Aal 35 84 Axmi173 ATX25337170.4 55% Cry32Aa1 36 85 Axmi174 ATX25337 143.6 63% Cry32Ba1 37 86Axmi175 ATX25337 146.0 54% Cry32Ca1 38 87 60% Axmi057 Axmi176 ATX25337143.0 54% Cry32Aa1 39 88 Axmi148 ATX13059 90.2 25% Cry3lAa1 40 89Axmi181 ATX12978 65.4 12% Cryl9Ba1 41 90 21% Axmi018 Axmi182 ATX1297859.0 13% Cry17Aa1 42 91

Example 13. Discovery of Novel Toxin Sequences from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 12 using the methods disclosed in Example 1.

TABLE 12 Cry homologues Molecular Nucleo- Amino Gene weight Closest tideSEQ acid SEQ name Strain (kD) homolog ID NO ID NO Axmi185 ATX13053 39.224% Cry55Aa1 43 92 Axmi186 ATX13053 39.9 21% Cry55Aa 44 93

Example 14. Discovery of Novel Toxin Sequences from Bacillusthuringiensis

Novel pesticidal genes were identified from the bacterial strains listedin Table 13 using the methods disclosed in Example 1.

TABLE 13 Cry homologues Molecular Nucleo- Amino Gene weight Closest tideSEQ acid SEQ name Strain (kD) homolog ID NO ID NO Axmi187 ATX12973 36.333% Cry15Aa1 45 94 (Bti) Axmi188 ATX12973 34.9 34% Cry15Aa1 46 95 (Bti)Axmi189 ATX12973 35.4 36% Cry15Aa1 47 96 (Bti)

Example 15. Expression in Bacillus

The toxin gene disclosed herein is amplified by PCR from pAX980, and thePCR product is cloned into the Bacillus expression vector pAX916, oranother suitable vector, by methods well known in the art. The resultingBacillus strain, containing the vector with axmi gene is cultured on aconventional growth media, such as CYS media (10 g/l Bacto-casitone; 3g/l yeast extract; 6 g/l KH₂PO₄; 14 g/l K₂HPO₄; 0.5 mM MgSO₄; 0.05 mMMnCl₂; 0.05 mM FeSO₄), until sporulation is evident by microscopicexamination. Samples are prepared and tested for activity in bioassays.

Example 16. Construction of Synthetic Sequences

In one aspect of the invention, synthetic toxin sequences weregenerated. These synthetic sequences have an altered DNA sequencerelative to the parent toxin sequence, and encode a protein that iscollinear with the parent toxin protein to which it corresponds, butlacks the C-terminal “crystal domain” present in many delta-endotoxinproteins. Synthetic sequences corresponding to the novel toxinsdisclosed herein are set forth in Table 14.

TABLE 14 Synthetic nucleotide sequences encoding toxins Wild-type GeneName Synthetic Gene Name SEQ ID NO: Axmi128 Axmi128bv01 97 Axmi128bv0298 Axmi130 Axmi130bv01 99 Axmi130bv02 100 Axmi131 Axmi131bv01 101Axmi131bv02 102 Axmi133 Axmi133bv01 103 Axmi133bv02 104 Axmi140Axmi140bv01 105 Axmi140bv02 106 Axmi141 Axmi141bv01 107 Axmi141bv02 108Axmi142 Axmi142bv01 109 Axmi142bv02 110 Axmi143 Axmi143bv01 111Axmi143bv02 112 Axmi144 Axmi144bv01 113 Axmi144bv02 114 Axmi146Axmi146bv01 115 Axmi146bv02 116 Axmi148 Axmi148_1bv01 117 Axmi148_1bv02118 Axmi148_2bv01 119 Axmi148_2bv02 120 Axmi149 Axmi149bv01 121Axmi149bv02 122 Axmi152 Axmi152bv01 123 Axmi152bv02 124 Axmi153Axmi153bv01 125 Axmi153bv02 126 Axmi154 Axmi154bv01 127 Axmi154bv02 128Axmi155 Axmi155bv01 129 Axmi155bv02 130 Axmi156 Axmi156_1bv01 131Axmi156_1bv02 132 Axmi156_2bv01 133 Axmi156_2bv02 134 Axmi156v03.04 197Axmi156v03.03 198 Axmi157 Axmi157bv01 135 Axmi157bv02 136 Axmi157v01.02199 Axmi157v01.03 200 Axmi157v01.04 201 Axmi158 Axmi158bv01 137Axmi158bv02 138 Axmi162 Axmi162bv01 139 Axmi162bv02 140 Axmi165Axmi165_1bv01 141 Axmi165_1bv02 142 Axmi165_2bv01 143 Axmi165_2bv02 144Axmi166 Axmi166bv01 145 Axmi166bv02 146 Axmi167 Axmi167bv01 147Axmi167bv02 148 Axmi168 Axmi168bv01 149 Axmi168bv02 150 Axmi169Axmi169bv01 151 Axmi169bv02 152 Axmi170 Axmi170bv01 153 Axmi170bv02 154Axmi171 Axmi171_1bv01 155 Axmi171_1bv02 156 Axmi171_2bv01 157Axmi171_2bv02 158 Axmi171v02.03 202 Axmi171v02.04 203 Axmi172Axmi172_1bv01 159 Axmi172_1bv02 160 Axmi172_2bv01 161 Axmi172_2bv02 162Axmi173 Axmi173_1bv01 163 Axmi173_1bv02 164 Axmi173_2bv01 165Axmi173_2bv02 166 Axmi174 Axmi174bv01 167 Axmi174bv02 168 Axmi175Axmi175bv01 169 Axmi175bv02 170 Axmi176 Axmi176_1bv01 171 Axmi176_ 1bv02172 Axmi176_2bv01 173 Axmi176_2bv02 174 Axmi177 Axmi177bv01 175Axmi177bv02 176 Axmi 178 Axmi178bv01 177 Axmi178bv02 178 Axmi179Axmi179bv01 179 Axmi179bv02 180 Axmi180 Axmi180bv01 181 Axmi180bv02 182Axmi181 Axmi18lbv01 183 Axmi181bv02 184 Axmi182 Axmi182bv01 185Axmi182bv02 186 Axmi185 Axmi185bv01 187 Axmi185bv02 188 Axmi186Axmi186bv01 189 Axmi186bv02 190 Axmi187 Axmi187bv01 191 Axmi187bv02 192Axmi188 Axmi188bv01 193 Axmi188bv02 194 Axmi189 Axmi189bv01 195Axmi189bv02 196

In another aspect of the invention, modified versions of synthetic genesare designed such that the resulting peptide is targeted to a plantorganelle, such as the endoplasmic reticulum or the apoplast. Peptidesequences known to result in targeting of fusion proteins to plantorganelles are known in the art. For example, the N-terminal region ofthe acid phosphatase gene from the White Lupin Lupinus albus (GenebankID GI:14276838; Miller et al. (2001) Plant Physiology 127: 594-606) isknown in the art to result in endoplasmic reticulum targeting ofheterologous proteins. If the resulting fusion protein also contains anendoplasmic retention sequence comprising the peptideN-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e. the “KDEL”motif (SEQ ID NO:208) at the C-terminus, the fusion protein will betargeted to the endoplasmic reticulum. If the fusion protein lacks anendoplasmic reticulum targeting sequence at the C-terminus, the proteinwill be targeted to the endoplasmic reticulum, but will ultimately besequestered in the apoplast.

Example 17. Bioassay of Axmi156 Gene Expression and Purification

The DNA regions encoding the toxin domain of Axmi156 was separatelycloned into an E. coli expression vector pMAL-C4x behind the malE genecoding for Maltose binding protein (MBP). These in-frame fusionsresulted in MBP-Axmi fusion proteins expression in E. coli. Forexpression in E. coli, BL21*DE3 was transformed with individualplasmids. A single colony was inoculated in LB supplemented withcarbenicillin and glucose, and grown overnight at 37° C. The followingday, fresh medium was inoculated with 1% of overnight culture and grownat 37° C. to logarithmic phase. Subsequently, cultures were induced with0.3 mM IPTG for overnight at 20° C. Each cell pellet was suspended in 20mM Tris-Cl buffer, pH 7.4+200 mM NaCl+1 mM DTT+ protease inhibitors andsonicated. Analysis by SDS-PAGE confirmed expression of fusion protein.

Total cell free extracts were run over amylose column attached to FPLCfor affinity purification of MBP-axmi fusion protein. Bound fusionprotein was eluted from the resin with 10 mM maltose solution. Purifiedfusion protein was then cleaved with either Factor Xa or trypsin toremove the amino terminal MBP tag from the Axmi protein. Cleavage andsolubility of the proteins was determined by SDS-PAGE.

Insect Bioassays

Cleaved proteins were tested in insect assays with appropriate controls.A 5-day read of the plates showed that the fusion protein cleaved withFactor Xa and trypsin had pesticidal activity against Diamondback mothand Southwestern cornborer pests.

Example 18. Activity of Axmi-171 on the Hemipteran Lygus hesperus

To test the ability of Axmi-171 to control plant pest species, the openreading frame was cloned into a vector designed to yield high levels ofsoluble protein by creating an N-terminal fusion between a protein ofinterest and a highly expressed, highly soluble protein. Axmi-171 wascloned into the expression vector pMal-C4x (New England Biolabs), as twodifferent constructs. The first construct (amxi-171.1, corresponding toSEQ ID NO:204, which encodes SEQ ID NO:205) did not yield high levels ofexpression and was not further pursued. Expression of the open readingframe constituting the internal methionine initiation codon(corresponding to SEQ ID NO:20, which encodes SEQ ID NO:69), yielded aconstruct designated pAX5557, whereby the shorter open reading frame wasfused in-frame to the maltose binding protein. The nucleotide sequenceof the construct is set forth in SEQ ID NO:206, and the encoded fusionprotein is set forth in SEQ ID NO:207. Using this construct, expressionof Axmi171.2 was induced as per manufacturer's instructions, and thefusion protein purified as known in the art.

Purified fusion protein was treated with a protease (Factor Xa, as knownin the art) to cleave the fusion and liberate Axmi171.2. Interestingly,Axmi171.2 was found to precipitate after this cleavage reaction.Precipitated protein was formed into a suspension in water, and testedin a bioassay on Lygus hesperus. Activity against Lygus heperus wasobserved, as shown in Table 15.

TABLE 15 Activity of resuspended Axmi 171.2 on Lygus hesperus SampleMortality (%) Axmi 171.2 Xa cut pellet, resuspended in water, 500 ppm 99± 2.5 Axmi 171.2 Xa cut pellet, resuspended in water, 200 ppm 98 ± 2.5Axmi 171.2 Xa cut pellet, resuspended in water, 100 ppm 88 ± 5   Axmi171.2 Xa cut pellet, resuspended in water, 50 ppm 80 ± 5   Water onlycontrol 0

Example 19. Activity of Axmi 171 on the Hemipteran Soybean Aphid (Aphisglycines)

To further test the ability of Axmi171 to control pests, cleavedpmal-Axmi-171.2 protein was tested in a bioassay on the soybean aphid(Aphis glycines), in a multi-container format. Multiple containers wereprepared containing either re-suspended Axmi171.2 protein (cleaved withFactor Xa as above), re-suspended 171.2 protein that had subsequentlybeen heated (100° C. for 30 minutes), or water control. Multiple animalswere exposed to the sample in each container, and at the end of theassay the number of containers with 100% mortality in each container wasrecorded. A score of zero was recorded for samples where none of thecontainers showed 100% mortality. A score of “1” was recorded forsamples where 0-25% of the containers showed 100% mortality, a score of“2” was recorded for samples where 25-50% of the containers had 100%mortality, a score of “3” was recorded where 50-75% of the containersshowed 100% mortality, and a score of “4” was recorded for samples where100% of the containers showed 100% mortality. Activity on this pest wasobserved compared to water only controls, as well as samples that hadbeen heated prior to inclusion in the sample.

Subsequently the Axmi171.2 open reading frame was cloned into a His-tagexpression vector (pRSF-1b; Novagen), and protein expressed and purifiedby virtue of the His tag (as known in the art) resulting in clonepAX5068. Soluble, purified Axmi171.2 protein was obtained. One samplewas heated to 100° C. for 30 minutes before being bioassayed. Assay ofAphis glycines in a multiple container format showed activity of theAxmi171.2 samples compared to controls.

Additionally, an E. coli expression clone expressing AXMI 171.2 proteinlacking an N-terminal tag or fusion was prepared, and denoted aspAX5069. A concentrated extract containing soluble Axmi171.2 protein wasprepared and similarly assayed for activity on Aphis glycines.

TABLE 16 Activity of resuspended Axmi 171.2 on Aphis glycines AverageSample Mortality Score Test 1: pAX5557 (purified pMal fusion) Axmi-171.2Fusion, Factor Xa cut; resuspended in water 2 Axmi-171.2 Fusion, FactorXa cut; resuspended in water and 0 heated Water control 0 Test 2:pAX5068 (purified His Tagged Protein) His-tagged, purified AXMI-171.2 1His-tagged, purified and heat-treated AXMI-171.2 0 Buffer (50 mM Tris(8.0) + 100 mM NaCl) 0 Test 3: pAX5069 (concentrated crude extract; noHis Tag) Untagged AXMI-171.2, concentrated extract 2 50 mM Tris (8.0) +100 mM NaCl 0

Example 20. Additional Assays for Pesticidal Activity

The ability of a pesticidal protein to act as a pesticide upon a pest isoften assessed in a number of ways. One way well known in the art is toperform a feeding assay. In such a feeding assay, one exposes the pestto a sample containing either compounds to be tested, or controlsamples. Often this is performed by placing the material to be tested,or a suitable dilution of such material, onto a material that the pestwill ingest, such as an artificial diet. The material to be tested maybe composed of a liquid, solid, or slurry. The material to be tested maybe placed upon the surface and then allowed to dry.

Alternatively, the material to be tested may be mixed with a moltenartificial diet, then dispensed into the assay chamber. The assaychamber may be, for example, a cup, a dish, or a well of a microtiterplate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson, J. L. & H. K. Preisler. 1992.Pesticide bioassays with arthropods. CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals “ArthropodManagement Tests” and “Journal of Economic Entomology” or by discussionwith members of the Entomological Society of America (ESA).

Example 21. Vectoring of the Toxin Genes of the Invention for PlantExpression

Each of the coding regions of the genes of the invention is connectedindependently with appropriate promoter and terminator sequences forexpression in plants. Such sequences are well known in the art and mayinclude the rice actin promoter or maize ubiquitin promoter forexpression in monocots, the Arabidopsis UBQ3 promoter or CaMV 35Spromoter for expression in dicots, and the nos or PinII terminators.Techniques for producing and confirming promoter—gene—terminatorconstructs also are well known in the art.

Example 22. Transformation of the Genes of the Invention into PlantCells by Agrobacterium-Mediated Transformation

Ears are collected 8-12 days after pollination. Embryos are isolatedfrom the ears, and those embryos 0.8-1.5 mm in size are used fortransformation. Embryos are plated scutellum side-up on a suitableincubation media, and incubated overnight at 25° C. in the dark.However, it is not necessary per se to incubate the embryos overnight.Embryos are contacted with an Agrobacterium strain containing theappropriate vectors for Ti plasmid mediated transfer for 5-10 min, andthen plated onto co-cultivation media for 3 days (25° C. in the dark).After co-cultivation, explants are transferred to recovery period mediafor five days (at 25° C. in the dark). Explants are incubated inselection media for up to eight weeks, depending on the nature andcharacteristics of the particular selection utilized. After theselection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated as known in the art.The resulting shoots are allowed to root on rooting media, and theresulting plants are transferred to nursery pots and propagated astransgenic plants.

Example 23. Transformation of Maize Cells with the Toxin Genes of theInvention

Maize ears are collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size are usedfor transformation. Embryos are plated scutellum side-up on a suitableincubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol;1.4 g/L L-Proline; 100 mg/L Casaminoacids; 50 g/L sucrose; 1 mL/L (of 1mg/mL Stock) 2,4-D), and incubated overnight at 25° C. in the dark.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to express the genes of the invention in plantcells are accelerated into plant tissue using an aerosol beamaccelerator, using conditions essentially as described in PCTPublication No. WO/0138514. After beaming, embryos are incubated for 30min on osmotic media, then placed onto incubation media overnight at 25°C. in the dark. To avoid unduly damaging beamed explants, they areincubated for at least 24 hours prior to transfer to recovery media.Embryos are then spread onto recovery period media, for 5 days, 25° C.in the dark, then transferred to a selection media. Explants areincubated in selection media for up to eight weeks, depending on thenature and characteristics of the particular selection utilized. Afterthe selection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated by methods known inthe art. The resulting shoots are allowed to root on rooting media, andthe resulting plants are transferred to nursery pots and propagated astransgenic plants.

Materials

DN62A5S Media Components per liter Source Chu'S N6 Basal 3.98 g/LPhytotechnology Labs Salt Mixture (Prod. No. C 416) Chu's N6 Vitamin 1mL/L (of 1000x Phytotechnology Labs Solution (Prod. Stock) No. C 149)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline  1.4 g/L Phytotechnology Labs Casaminoacids 100 mg/L FisherScientific Sucrose   50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1 mL/L(of 1 mg/mL Stock) Sigma D-7299)

Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N KCl, addGelrite (Sigma) to 3 g/L, and autoclave. After cooling to 50° C., add 2ml/L of a 5 mg/ml stock solution of Silver Nitrate (PhytotechnologyLabs). Recipe yields about 20 plates.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1-18. (canceled)
 19. A recombinant polypeptide with pesticidal activity,selected from the group consisting of: a) a polypeptide comprising theamino acid sequence of any of SEQ ID NO:94, 95, and 96; b) a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of any of SEQ ID NO:94, 95, and 96; and c) apolypeptide that is encoded by any of SEQ ID NO:45, 46, and
 47. 20. Therecombinant polypeptide of claim 19, further comprising heterologousamino acid sequences.
 21. A composition comprising the recombinantpolypeptide of claim
 19. 22. The composition of claim 21, wherein thecomposition is selected from the group consisting of a powder, dust,pellet, granule, spray, emulsion, colloid, and solution.
 23. Thecomposition of claim 21, wherein the composition is prepared bydesiccation, lyophilization, homogenization, extraction, filtration,centrifugation, sedimentation, or concentration of a culture ofbacterial cells.
 24. The composition of claim 21, comprising from about1% to about 99% by weight of the recombinant polypeptide.
 25. A methodfor controlling a lepidopteran, coleopteran, nematode, or dipteran pestpopulation, comprising contacting the population with apesticidally-effective amount of the recombinant polypeptide of claim19.
 26. A method for killing a lepidopteran, coleopteran, nematode, ordipteran pest, comprising contacting the pest with, or feeding to thepest, a pesticidally-effective amount of the polypeptide of claim 19.27. A method for producing a polypeptide with pesticidal activity,comprising culturing a host cell comprising a nucleic acid moleculeencoding the recombinant polypeptide of claim 19 under conditions inwhich the nucleic acid molecule encoding the recombinant polypeptide isexpressed.
 28. A plant comprising the recombinant polypeptide of claim19.
 29. The plant of claim 28, wherein the plant is a plant cell.